1. Field of the Invention
Generally, this application relates to molecular biology and microbiology chemistry processes and apparatuses including optical measuring or testing means. Certain embodiments relate to devices, systems, and methods for imaging assays exhibiting chemiluminescence or other luminescence.
2. Background
Detecting and identifying certain biomolecules can be important in studying biological systems, such as plants and animals. Such systems may exhibit only small amounts of a biomolecule, such as a protein, which in some instances may only be accurately measured by high-end, laboratory-grade equipment and processes. Many assay techniques have been developed over the last decades for accurately acquiring such measurements.
A variety of assays use enhanced chemiluminescence (CL) to detect small quantities of a biomolecule. Techniques using an Enzyme-linked Immunosorbent Assay (ELISA) or a Western Blot are often used to detect low abundance proteins.
Chemiluminescence techniques differ from fluorescence in that no outside excitation light is needed. Therefore, there is virtually no light emission from areas of the assay where the chemiluminescence reaction is not happening, and thus there exists a very low optical background. This is one of the primary reasons for the ability to detect low amounts of optical emissions in CL assays.
Chemiluminescence emitted optical signals are typically characterized by: being 1) weak and 2) non-constant over time. Weakness of the optical signals drives the need for high signal-to-noise detection methods. The use of high-efficiency imaging optics and extremely low noise detectors are taught in the art for measuring chemiluminescence. The non-constant nature of light emission of typical chemiluminescence substrates naturally implies using whole-image capturing methods. Such imaging allow for collecting equal amounts of light from all emitting areas of the assay at the same time and over long integration times.
In the past, one of the most common methods for capturing optical emissions from a CL assay was the use of photographic film. One would expose a photographic film in close proximity to a light-emitting Western Blot, for example. However, there are a number of limitations to using film. Oftentimes, film exhibits a non-linear response to impinging photons and has limited dynamic range. There is also the need for careful handling of undeveloped film and special dark-room facilities and chemicals to develop and process the film.
Furthermore, there is often a need to digitally capture an image of the assay in order to store it for future reference. To accomplish this, film users often capture an image of developed film using a digital camera or a flat-bed scanner. This approach requires additional imaging equipment, can be non-quantitative, and is prone to errors in image capture and reproduction.
Directly imaging the optical emissions of a CL assay using a digital, Charge-Coupled Device (CCD) camera can overcome many of the difficulties associated with using photographic film. CCD cameras provide a linear response to impinging photons over a wide dynamic range, no chemicals are needed for developing, and digital data can be read directly from the digital camera and easily stored for further analysis and quantification. Exposing a high-quality, cryogenically-cooled CCD array to a Western Blot over a long period of time in a very dark room has been taught in the art. A number of such CCD-based imagers exist on the market nowadays that achieve sensitivity that is comparable to film. An example of a CCD-based imager is the LI-COR Odyssey® Fc imager. The CCD-based imagers have deeply cooled chips that are highly sensitive to light.
Complementary Metal Oxide Semiconductor (CMOS) image sensors have been developed in parallel with CCD sensors. Unlike those for CCDs, each pixel in a CMOS image sensor has its own charge-to-voltage conversion components. This can result in lower uniformity than CCD image sensors. However, as lithographic and other manufacturing processes improve, CMOS image sensor uniformity has become less of an issue. As the technology improves, CMOS image sensor arrays may be used in place of CCD image sensor arrays in many markets.
There exists a need in the art for a small, inexpensive, digital alternative to CL imaging by film that attains the same or better sensitivity.